Process for making cheese



United States Patent 3,212,905 PROCESS FOR MAKING CHEESE Kei Arima,Komagome, Bunkyo-ko, Tokyo, and Shinjiro Iwasaki, Urawa-shi, Japan,assignors to Meito Sangyo abushiki Kaisha, Nagoya, Japan, a corporationof apan No Drawing. Original application Jan. 4, 1962, Ser. No. 164,393,now Patent No. 3,151,039, dated Sept. 29, 1964. Divided and thisapplication June 25, 1964, Ser. No. 387,256

Claims priority, application Japan, July 20, 1961, 36/25,664 5 Claims.(Cl. 99-116) This is a division of application Serial No. 164,393, filedJanuary 4, 1962, and now US. Patent No. 3,151,039.

This invention relates to enzyme produced by fungi. More particularly,it relates to a novel milk coagulating enzyme microbial rennet obtainedfrom the cultures of fungi selected from the group consisting of Mucorpusillus Lindt of genus Mucor, genus Rhizopus, genus Monascus, genusAscochyta, genus Sclerotium, genus Colletotrichum, their varieties, andthe natural and artificially induced mutants thereof which possess mostof the characteristic properties of the parent fungi concerned; and themethod of preparing the same. Additionally, the invention relates to amethod of making cheese by using such as enzyme.

In the description to be given hereinafter the milk coagulating enzymeobtained by the cultivation of the aforementioned fungi will be referredto as microbial rennet.

Heretofore, as a milk coagulating enzyme the socalled rennet that isobtained from the fourth stomach of sucking calves is well-known. Sincethis rennet is powerful in so far as its milk coagulating activity isconcerned, it has been used widely as an enzyme indispensable for cheesemaking, and is still being used. However, inasmuch as the raw materialused is the fourth stomach of sucking calves, there is a limit to thesupply of raw material, and furthermore it is costly.

Generally, proteases coagulate milk. Hence, when viewed from thestandpoint of coagulating activity the proteases such as those of animalorigin, namely pepsin, trypsin, chymotrypsin, cathepsin, etc. and thoseof vegetable origin, namely, papain, ficin, bromelain, etc. all havemilk coagulating activity to a certain degree. Also the coagulatingactivity of the proteases that are produced by bacteria such as, forexample, Pseudomonas fluorescens, Bacillus subtilfs and Serratiamarcescens are already known. However, these proteases having highprotein digesting activity and low protein coagulating activity, theycannot be put to practical use as milk coagulating enzymes.

Furthermore, as an enzyme produced from the cultures of fungi, there isalso known a coagulating or curding enzyme that can be produced bycultivating specifically the fungus Mucor nouxii upon a suitable culturemedium, preferably on rice. However, it is not known at all that bycultivating the fungus selected from the group as particularly specifiedin the present invention, enzyme having high milk coagulating activitybut very low protease activity can be obtained in high yield in themedium, and that using thus obtained enzyme cheese can be madeadvantageously on a commercial scale.

In consequence of having furthered our studies concerning the productsproduced by the culture of microbes, fungi in particular, we found thatit is possible to produce on a commercial scale and at low cost a novelmilk coagulating enzyme microbial rennet having very low proteaseactivity but remarkable high milk coagulat- 3,212,905 Patented Oct. 19,1965 ing activity by utilizing those fungi, their varieties and thenatural and artificially induced mutants thereof that possess most ofthe characteristic properties of the parent fungi concerned, whichhitherto were considered as being unable to produce milk coagulatingenzymes of any practical value; and that by using the microbial rennetit is possible to make cheese advantageously on a commercial scale.

Accordingly, the primary object of the present invention is to provide anovel milk coagulating enzyme microbial rennet having very low proteaseactivity but remarkably high milk coagulating activity by cultivatingfungus selected from the group consisting of Mucor pusillus Lindtbelonging to the genus Mucor, genus Rhizopus, genus Monascus, genusAscochyta, genus Sclerotium, genus Colletotrichum, their varieties andthe natural and artificially induced mutants thereof that possess mostof the characteristic properties of the parent fungi concerned.

It is another object of the invention to provide a method of producingmicrobial rennet by cultivating on a suitable medium a fungus selectedfrom the group as mentioned hereinabove.

A still another object of the invention is to provide a method ofcollecting microbial rennet from the medium and preparing microbialrennet into its finished form.

A further object of the invention is to provide a method of makingcheese using this microbial rennet.

Other objects and advantages of the invention will be apparent from thefollowing detailed description thereof.

According to our studies, the fungi that are capable of producing thenovel milk coagulating enzyme, microbial rennet, having very lowprotease activity but remarkably high milk coagulating activity includethe fungi selected from the group consisting of Muoor pusz'llus Lindtbelonging to the genus Mucor, genus Rhizopus, genus Monascus, genusAscochyta, genus Sclerotium, genus Colletotrichum, their varieties, andthe natural and artificially induced mutants thereof having most of thecharacteristic properties of the parent fungi concerned.

As already described, that Mucor rouxii produces upon a suitable culturemedium, preferably on rice, a coagulating or curding enzyme is known.However, it is not known at all particularly that Mucor pusillus Lindtproduces a milk coagulating enzyme having very low protease activity butremarkably high milk coagulating activity in high yield in the medium.Furthermore the genus Mucor covering a great number of species therein,it was found that many of the species do not produce enzymes possessingthe desired milk coagulating activity. Moreover, Mucor pusillus Lindtclearly exhibits different characteristic properties from that of Mucorrouxii. On a Koji agar slant culture at 30 C., Mucor pusillus Lindtexhibits the characteristic properties as described hereinafter: Namely,the colony which is white at first, upon the formation of sporangiabecomes gray or grayish-brown, the height of colony being less than 2mm. The sporangiophores are at first transparent and colorless and donot branch, but subsequently branch with formation of sporangia on eachof the tips thereof. The sporangiophores which are 5-20/L1I1 width, uponmaturity, become grayish in color, and in most instances the formationof septa occur near the tips. The sporangia have a lateral diameter of40-90 and are of either gray or grayish-brown color. The columellae arelight brown in color and are either eggor pear-shaped havinglongitudinal diameters of 10-50 The sporangiospores are spherical withdiameters of 25-411.. Neither chlamydospore nor zygospore is observed.While the description of those cultivated under other culture conditionswill be omitted, those that have been cultivated by means of a culturemedium such as malt agar, potato agar, possess properties that aresubstantially the same. When the chief points of diiference between thecharacteristic properties of Mucor pusillus Lindt used in the presentinvention and that of Mucor 4 itself will vary depending upon theadaptability of the fungus that is used for the production of microbialrennet.

While the temperature of cultivation will vary depending on the fungusemployed, usually between about 20 rouxii are set forth, they are asshown in the following 5 40 C. is suitable. It is usual to carry out thecultivation Table I. for about 2-7 days by means of liquid culture orsolid TABLE I Characteristic Properties Sporangiospore Growth FungusEehghn of S Oran mm Chlamydospore T erip. (mm) p g Shape Diameter I) M.pusillus Llndt 2 Formed well Spherical 2.54.-.. Not f0rrned -50. M- ml 4Rarely formed Eggshaped or 4-5 Numerous for- 945.

spheroidal. mations.

According to this invention the fungi selected from the culture at TParheularly and Prefehebiy group consisting of the genus Rhizopus, genusM 0 nascu S, under aeroblc conditions. Thus, while 1n case of liquidgenus Ascochyta, genus Sclerotium, genus Colletotrichum, culture.surfaee culture may employed better results their varieties, and thenatural and artificially induced are ebtamed by means of i culture.Submerged mutants thereof having most of the characteristic properh Asafesult of h e t f? above the ties of the parent fungi concerned canalso be used as the i eeagelahhg enzyme mlereblal h e Produce: fungi forproducing microbial rennet As fungi belong in the medium. In order tocollect the m croblal rennet ing to the foregoing fungi there can benamed Rhizopus from e culture h m e of Sohd culture It IS delemar,Rhizopus niveus, Rhizopus nodesus, Rhizopus m l y extractmg w water, fan aqueous pseudochinensis, Rhizopus Peka II, Rhizopus Peka I, solutlonwhlle m of hquld 9 It 18 2? by h Rhizopus usamii, Rhizopus batatas,Rhizopus thermosus, f the .myeehum .Whereby m both cases mleroblalMonascus anka, Ascochyta visa, Sclerotium oryzae-sativa, rennet contammgSolutlon can be collected flquecus Colleto richum atramentarium, etc. Asthese fungi eX- solvents can hemmed e eontammg salts tend over a broadrange, an enumeration of the propersllch as e h i petasslum h ties ofeach will be omitted. However, in Genera of trons containing acids suchas hydrochloric acid, citric acid, Fungi, by Frederic E Clements andCornelius Shear, acetic acld, etc. as well as various other types ofbuffer published by Hafner Publishing Co., New York, 1957, Solutlens'there is described concerning the characteristic proper- Thus eetamedmler'eblel P? contamlhg Sohmon ties of the genus Rhizopus, genusMonascus, genus Ascoean.then. be concentrated or l out h chyta, genusSclerotinm, and genus Colletotrichum to 40 purrficatlon treatments suchas precrpitatlon with organlc which genera the foregoing fungi belong.solvents, salting out, concentrat on under reduced pres- NeXt, will bedescribed the method according to the Purification by means 9. exchange?AS invention, by which a fungus Selected from the group precipitatingsolvents, water miscible orgamc solvents Sisting of Manor past-"us Lindtbelonging to genus Mncon such as methanol, ethanol, rsopropanol,acetone, etc. can genus Rhizopus, genus Monascus, genus Ascochyta, genushe e And as the e h out agents any of the salts Sclerotium, and genusColletotrichum, their varieties, and that dlseolve very Raddy In Watermay be e Such as the natural and artifically induced mutants thereofthat ammehhhh Sulfate sodhlm Sulfate magheslhm Sulfate possess most ofthe characteristic properties of the parent fungi concerned iscultivated, thereby effecting the pro- Mleheblal e hovel mhk eoegulahhgh h' dncnon of microbial rennet in its medium, then accordmg to thepresent mvention, exhibits characteristics centrating and preparing thesame. What is referred to as that are elehrly dlfiereht from hhltherto'khowh the concentrating, obtaining and preparing of microbialteases of animal or vegetable orlgrn or those proteases rennet in theinvention denotes the process of obtaining that h predueed byotherhhcrobes' microbial rennet in a still more refinable state than in itsMlerehlal Iehhet possesses P mhk coagulat' raw material state or in astate in which it is of a higher g achvlty h Its h h h very Weak' hconcentration or higher Purity According to the inven ITllllCcoagulating activity of microbial rennet, likewlse tion the medium to beemployed for cultivating the fungi, as in the mstance of rennet obtainedfrom the fourth as to its composition may be any suitable artificial orstomach of sucking calves, shows powerful activity in the natural mediumso long as it contains at least a carbon h F as h PH rises its Potencydecreases: no source, a nitrogen source and inorganic salts. As thecaraehvlty hemg hmhlfested when e PH in the alkaline bon source, themonosaccharides, disaccharides and polysldee makme cheese mlcroblalrennet can be saccharides which are assimilable by the fungi, such asused as m ccnvenhohal h sucrose, lactose, glucose, starch, etc. can beused. Simi- Wheh curds are h the mtrogeh that transfers into larly inCase of the nitrogen Source so long as they are the wheyafter 150minutes was about 20% determined assimilable, a wide range of nitrogensources can be used by h Kleldahl method h smee it resembles such asinorganic ammonium salts, amino acids, and a stahtlauy h mStanFSFSmtmgfrom the i of rennet wide variety of other protein substances, etc. Theinthe eaeem m e .mllk l e ,h when organic Salts may also includg, forexample p p hhe tphrotihise activity} 1 of m croblal rennet was measuredmagnesium salts, calcium salts, and numerous other ini s f i organicsalts. In any event, the carbon or nitrogen source h h lg 22 f m1 i f.achvlty the of the natural or artificial medium may be of any kind soproduced by i; i ij 8 g protease long a itisutilizable bythe fungus tobe employed As a rennet, 0.059 0.1). while that of mierobial 11 16?Preferred Inedlum that of Wheat bran y be giv It is of the presentinvention was less than 0.102 (O.D.), into be understood, Of course,that the amounts of carbon dicating that this enzyme was a completelydifferent type d n trogen sources and the composition of the medium ofenzyme than the usual protease, its milk coagulating 5 activity beingunusually powerful as compared with its protease activity. Consequently,it is befitting to be referred to as microbial rennet.

Since microbial rennet, which is cultivated, collected and prepared asdescribed hereinabove, is obtained by cultivating microbes, it ispossible to mass-produce it very profitably from the economic standpointon a commercial scale. Hence, while it is widely utilizable for makingdesserts, ice cream and for other uses such as preparation ofconfectionaries, it is particularly of great advantage in connectionwith cheese making.

In making cheese the step of producing a curd by adding to milk anenzyme to coagulate it is a necessary step for making any type ofcheese. As the milk coagulating enzyme, rennet, which has already beenmentioned, is well known, and as its milk coagulating activity ispowerful, it is considered to be indispensable for the making of cheese.However, according to this invention, it is possible to provide cheeseproducts of excellent quality very readily on a commercial scale withoutthe employment of rennet.

In order to more clearly understand the present invention, severalexamples illustrating the mode of practicing the invention are givenbelow, it being understood that the same are merely intended in anillustrative sense, and the invention should not be limited thereby, itbeing possible to make various changes as to the fungi employed, thecomposition of the medium, the method of collecting, etc. withoutdeparting from the spirit and scope of the invention.

Example 1 To 10 parts of wheat bran 7 parts of water was added thereon.After mixing, it was cooked for 30 minutes at 100 C. This was inoculatedwith a strain belonging to Mucor pusillus Lindt, and was incubated at 30C., whereby on the 3rd day the milk coagulating activity reached itsmaximum. The wheat bran to which water was added in the proportion of 10grams of the former to 50 ml. of the latter was allowed to stand at roomtemperature for 1 hour, after which the mycelium was separated byfiltration and 40 ml. of microbial rennet solution was obtained. Themilk coagulating activity of this extract was 800 Soxhlet units/ml. fora 10% solution of powdered skimmed milk containing A M of CaClConsequently, from 10 grams of wheat bran were ob tained 32,000 units ofmicrobial rennet. Next, by salting out this extract with ammoniumsulfate of 0.22-0.66 saturation, practically all of its activity couldbe transferred to the precipitate portion. Furthermore, activityrecoveries of 78%, 80%, 60% and 75% were shown respectively by means oforganic solvents of the following concentrations: namely, ethanol 70%,methanol 75%, acetone 66% and isopropanol 70%. The activity of microbialrennet thus obtained was 100 Soxhlet units/ mg, While that of the rennettablets of Chr. Hansens Laboratory, Inc., was 200 Soxhlet units/mg.

Then microbial rennet of the invention was used and cheese was made.Namely, to 55 kg. of raw milk mixed with 15 kg. of skimmed milk whichwere maintained at 30 C. were added 13 grams of KNO followed by 8.5grams of CaCl -2H O and 1 kg. of starter. Ten minutes later, 5 grams ofmicrobial rennet obtained as described above was added, and the mixturewas stirred. After 25 minutes standing, the curd was formed and cuttingthereof was performed. Cooking was carried out in three consecutivesteps at 33 C., 36 C. and 38 C., and after 1 hour and 20 minutes ofcooking at 38 C., 120 grams of sodium chloride was added. The drainingof the whey from the curd was normal, and the texture of the curd wasfine and smooth. Thus was obtained 6.3 kg. (yield 8.9%) of green cheese.

Example 2 A medium containing of powdered skimmed milk, 1% of glucose,0.1% of yeast extract, M of CaCl was inoculated with a strain belongingto Mucor pusillus Lindt, and after 4 days shaking culture at 30 C., themycelium was separated by filtration, and an enzyme solution wasobtained. The activity of this enzyme solution was Soxhlet units/ml.Thus 1 ml. of it was equivalent to 0.05 mg. of rennet tablets. 'Fromthis enzyme solution by a similar method as in Example 1 a purifiedsolid enzyme preparation was obtained. Next, to 53 kg. of raw milk mixedwith 17 kg. of skimmed milk which were maintained at 30 C. were added 13grams of KNO followed by 8.5 grams of CaCl -2H O and 1 kg. of starter.Ten minutes later, 5.5 grams of microbial rennet obtained as describedabove was added, and the same operations as in Example 1 were performedwhereby was obtained 6.2 kg. of green cheese.

Example 3 The wheat bran medium described in Example 1 was inoculatedwith a strain belonging to Rhizopus Peka II and incubated for 4 days at30 C., following which extraction was performed as in Example 1, theenzyme solution so obtained showing 380 Soxhlet units/ml. On the otherhand, when a 10% powdered skimmed milk solution containing 1% of glucoseand M of CaCl was inocluated with this strain, shaking culture performedfor 3 days at 30 C. followed by separating the mycelium by filtration,the enzyme solution obtained showed 70 Soxhlet units/ml. By means of themethod described in Example 1 microbial rennet solution was precipitatedand solidified. Using these preparations and carrying out the sameprocess as in Example 1, cheese was obtained.

Example 4 A medium identical to that described in Example 2 wasinoculated with a strain belonging to Monascus anka, and after 6 daysshaking culture at 30 C., the mycelium was separated by filtrationwhereby was obtained an enzyme solution. The activity of this enzymesolution was 68 Soxhlet units/ml. Powdered microbial rennet was obtainedfrom this enzyme solution by the same method as in Example 1. Using thisenzyme preparation and carrying out the same process as in Example 1,cheese was obtained.

Example 5 A medium prepared by cooking and extracting 200 grams ofpotatoes in 1 liter of Water followed by the addition of 2% of glucoseand 0.1% of yeast extract was inoculated with a strain belonging toAscochyta visa, and after cultivating for 4 days at 30 C. the enzymesolution obtained by separation of the mycelium by filtration showed anactivity of 70 Soxhlet units/ml. This solution was treated as in Example1 whereby powdered microbial rennet was obtained. This enzyme was used,and by processing as in Example 1 cheese was obtained.

Example 6 10 parts of wheat bran was mixed with 7 parts of watercontaining 0.5% of peptone, 0.1% of yeast extract and 1% of glucose.After cooking this mixture, it was inoculated with a strain belonging toSclerotium oryzaesativa and cultivated for 5 days at 28 C., followed byextraction as in Example 1. The enzyme solution thus obtained showed anactivity of 70 Soxhlet units/ml. From this solution by the same methodas in Example 1, powdered microbial rennet was obtained. Using thispowdered enzyme and processing as in Example 1, cheese was obtained.

Example 7 The same medium as described in Example 5 was inoculated witha strain belonging to Co lletotrz'chum atramemarium. After cultivatingfor 4 days at 30 C., the mycelium was separated by filtration wherebywas obtained an enzyme solution showing an activity of 59 Soxhletunits/ml. From this solution powdered microbial rennet was obtained bythe same method as described in Example 1. Using this powdered enzyme,cheese was obtained by processing as in Example 1.

Having thus described the nature of the invention, What is claimed is:

1. A method of making cheese which comprises cultivating on a suitablemedium a fungus of Mucor pusillus Lindt, collecting as the chief producta milk coagulating enzyme, incorporating said enzyme in the materialmilk, and effecting the coagulation of said mixture.

2. A method in accordance with claim 1 wherein said medium ischaracterized by forming an aqueous solution having a pH in the rangefrom about 3 to about 8.

cultivating is carried out under aerobic conditions.

5. A method in accordance With claim 2 wherein said cultivating iscarried out at a temperature in the range of from about 20 C. to about40 C.

References Cited by the Examiner UNITED STATES PATENTS 9/21 Takamine 99116 A. LOUIS MONACELL, Primary Examiner.

1. A METHOD OF MAKING CHEESE WHICH COMPRISES CULTIVATING ON A SUITABLEMEDIUM A FUNGUS OF MUCOR PUSILLUS LINDT, COLLECTING AS THE CHIEF PRODUCTA MILK COAGULATING ENZYME, INCORPORATING SAID ENZYME IN THE MATERIALMILK, AND EFFECTING THE COAGULATION OF SAID MIXTURE.